Drug repurposing through Biophysical Insights: Focus on Indoleamine 2,3-Dioxygenase and Tryptophan 2,3-Dioxygenase Dual Inhibitors.

Introduction: TDO (tryptophan 2,3-dioxygenase) and IDO (indoleamine 2,3-dioxygenase) are structurally distinct enzymes that catalyse the oxidation of tryptophan (Trp) leading to local Trp depletion and formation of immunesuppressive kynurenine pathway (KP) metabolites and dampening of the immune response in the tumour environment. IDO is known to be up-regulated in numerous cancers. Recently, TDO was shown to be up-regulated in various tumour types. Hence, selective IDO inhibitors may have a limited effect in cancers harbouring both TDO and IDO. To test this hypothesis we utilised recently identified proprietary dual-active and highly selective TDO/IDO inhibitors to modulate kynurenine (Kyn) levels in glioma cancer cells expressing either TDO alone or expressing both TDO and IDO. In addition, we measured Kyn modulation in human peripheral blood mononuclear cells (PBMCs) following exposure to dual and selective inhibitors. Methods: Assays were established to measure the effects of dual-active and highly selective TDO/IDO inhibitors on Kyn production in 1) human A172 glioma cells naturally expressing TDO alone or expressing both TDO and IDO following IDO induction using Interferon γ (IFNΓ) and in 2) freshly isolated human PBMCs treated with IFNΓ. Results: Dual-active and selective TDO/IDO inhibitors demonstrated distinct Kyn modulating activities in A172 glioma cells expressing either TDO alone or both TDO and IDO. Dual-active molecules fully ablated Kyn levels in both TDO-expressing and TDO/IDO-expressing cells. Crucially, selective inhibitors were unable to fully block Kyn in A172 cells co-expressing both enzymes. As expected TDO-selective inhibitors were highly active in A172 cells expressing TDO alone. IDO-selective inhibitors were largely inactive in A172 cells naturally expressing TDO alone. However, they gained significant activity upon induction of IDO in these cells following IFNΓ exposure. Interestingly, TDO-selective inhibitors were unable to modulate Kyn levels in PBMCs whereas IDO-selective and dual-active molecules were able to potently block Kyn production. Discussion: These data demonstrate that dual-active TDO/IDO inhibitors are most effective at reducing Kyn levels in cancer cells co-expressing TDO and IDO. In addition, the data suggest that IFNΓ-treated PBMCs primarily express IDO with no measurable Kyn production from TDO. This study demonstrates the utility of applying highly selective and dual active inhibitors of these Trp-catabolizing enzymes as probes for defining the Kyn-producing component from PBMCs. In summary, blocking both TDO and IDO activity with dual-active inhibitors maybe complementary rather than redundant in cancers harbouring both of these enzymes; and IDO appears to be the primary enzyme driving Trp metabolism in PBMCs. Citation Format: Alan Wise, Barry E. McGuinness, Sarah C. Trewick, Phillip M. Cowley, Nicola Bevan, Clare Szybut, Thomas J. Brown. In vitro kynurenine modulation by novel dual-acting and selective tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO) inhibitors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4292. doi:10.1158/1538-7445.AM2015-4292

Exposure to high sugar diet (HSD) serves as an experimental model of insulin resistance (IR) and type 2 diabetes (T2D) in mammals and insects. Peripheral IR induced by HSD delays emergence of pupae from larvae and decreases body weight of Drosophila imago. Understanding of mechanisms of IR/T2D is essential for refining T2D prevention and treatment strategies. Dysregulation of tryptophan (TRP) - kynurenine (KYN) pathway was suggested as one of the mechanisms of IR development. Rate-limiting enzyme of TRP - KYN pathway in Drosophila is TRP 2,3-dioxygenase (TDO), an evolutionary conserved ortholog of human TDO. In insects TDO is encoded by vermilion gene. TDO is not active in vermilion mutants. In order to evaluate the possible impact of deficient formation of KYN from TRP on the inducement of IR by HSD, we compared the effect of HSD in wild type (Oregon) and vermilion mutants of Drosophila melanogaster by assessing the time of white pupae emergence from larva and body weight of imago. Delay of emergence of pupae from larvae induced by high sucrose diet was less pronounced in vermilion (1.4 days) than in Oregon flies (3.3 days) in comparison with flies maintained on standard diet. Exposure to high sucrose diet decreased body weight of Oregon (but not vermilion) imago. Attenuation of high sucrose diet-induced IR/T2D in vermilion flies might depend on deficiency of TRP - KYN pathway. Besides IR/T2D, HSD induces obesity in Drosophila. Future studies of HSD-induced obesity and IR/T2D in TDO deficient vermilion mutants of Drosophila might help to understand the mechanisms of high association between IR/T2D and obesity. Modulation of TRP - KYN metabolism might be utilized for prevention and treatment of IR/T2D.

Expanding the Therapeutic World of Tryptophan Metabolism.

Kynurenine pathway enzyme KMO in cancer progression: A tip of the Iceberg

IDO1/TDO dual inhibitor RY103 targets Kyn-AhR pathway and exhibits preclinical efficacy on pancreatic cancer.

Alzheimer’s disease (AD) is associated with progressive endogenous neurotoxicity and hampered inflammatory regulation. The kynurenine (Kyn) pathway, which is controlled by tryptophan 2,3-dioxygenase (TDO), produces neuroactive and anti-inflammatory metabolites. Age-related Kyn pathway activation might contribute to AD pathology in humans, and inhibition of TDO was found to reduce AD-related cellular toxicity and behavioral deficits in animal models. To further explore the effect of aging on the Kyn pathway in the context of AD, we analyzed Kyn metabolite profiles in serum and brain tissue of the APP23 amyloidosis mouse model. We found that aging had genotype-independent effects on Kyn metabolite profiles in serum, cortex, hippocampus and cerebellum, whereas serum concentrations of many Kyn metabolites were reduced in APP23 mice. Next, to further establish the role of TDO in AD-related behavioral deficits, we investigated the effect of long-term pharmacological TDO inhibition on cognitive performance in APP23 mice. Our results indicated that TDO inhibition reversed recognition memory deficits without producing measurable changes in cerebral Kyn metabolites. TDO inhibition did not affect spatial learning and memory or anxiety-related behavior. These data indicate that age-related Kyn pathway activation is not specific for humans and could represent a cross-species phenotype of aging. These data warrant further investigation on the role of peripheral Kyn pathway disturbances and cerebral TDO activity in AD pathophysiology.

Parallel discovery of selective and dual inhibitors of tryptophan dioxygenases IDO1 and TDO2 with a newly-modified enzymatic assay

Tryptophan catabolism through the kynurenine pathway produces the oncometabolite kynurenine, which is strongly implicated in cancers such as triple-negative breast cancer (TNBC). The enzymes indoleamine 2,3-dioxygenase (IDO1) and tryptophan 2,3-dioxygenase (TDO) drive this pathway and promote an immunosuppressive tumour microenvironment, making them an attractive therapeutic target. However, no approved drug currently inhibits both enzymes simultaneously. In this study, we employed a machine learning (ML)-driven virtual screening pipeline to identify potent dual IDO1 and TDO inhibitors. Initially, an in-house ML classification model was developed using IC50 values from 1,037 distinct dual inhibitors sourced from the ChEMBL and BindingDB databases. Among the various models evaluated, the eXtreme Gradient Boosting with Random Forest (XGBRF) classifier achieved the highest performance (95% accuracy) and was selected to screen the MEGxp database. Subsequent molecular docking, MM-GBSA calculations, rescoring, and ADMET profiling identified two promising candidates, NP000319 and NP003833. Both compounds also showed predicted anticancer potential against MDA-MB-231 TNBC cells. Furthermore, the stability of the protein-ligand complexes was confirmed through 100 ns molecular dynamics simulations. Overall, the study highlights the value of ML-driven dual-inhibition strategies and provides strong leads for future experimental validation and potential therapeutic development for TNBC.

Effect of water-immersion restraint stress on tryptophan catabolism through the kynurenine pathway in rat tissues.

Comparative effects of oxygen on indoleamine 2,3-dioxygenase and tryptophan 2,3-dioxygenase of the kynurenine pathway

e14228 Background: Kynurenine production by indoleamine 2,3-dioxygenase (IDO) is critical for tumor immune suppression through effector T cell anergy and regulatory T cell proliferation. This has led to the rapid development of IDO inhibitors for cancer immunotherapy. However, results from recent clinical trials have been disappointing and this is partly due to pathway redundancy. Tryptophan 2,3-dioxygenase (TDO), another important enzyme of the kynurenine pathway, plays a compensatory role in the absence of IDO activity. Therefore, we developed a dual inhibitor of IDO and TDO to achieve maximal inhibition of the kynurenine pathway and alleviate tumor immune suppression. Methods: Small-molecule inhibitors of IDO and TDO were synthesized and evaluated using in vitro cell-based assays. Pharmacokinetic and pharmacodynamic profiles were assessed for these inhibitors. Tumor-bearing mice were treated with CMG017 per os, either alone or in combination with immune checkpoint inhibitors (ICIs). The tumor microenvironment (TME) was assessed through histological, flow-cytometric, and Nanostring immune profiling analyses. Results: CMG017 suppressed kynurenine production more effectively than inhibitors targeting either IDO or TDO alone, in various human and murine cancer cell lines. Single administration of CMG017 showed favorable pharmacokinetic profiles compared with an IDO1 selective inhibitor. Repeated once-daily per os administration of CMG017 decreased kynurenine concentration in both tumors and plasma of tumor-bearing mice and delayed tumor growth without significant toxicity. CMG017 induced dramatic changes in immune-related genes in TME and enhanced intratumoral infiltration of CD8+ effector T cells. The anti-tumor activity of CMG017 was almost negated when T cells were depleted, indicating the importance of adaptive immunity for the in vivo efficacy of CMG017. Of note, combination immunotherapy of CMG017 with ICIs (anti-PD-1 and anti-CTLA-4) led to durable tumor regression and long-term overall survival. Mice with complete tumor regression were immune to tumor re-challenge, indicating the establishment of immunological memory. Conclusions: CMG017, a dual inhibitor of IDO and TDO, potently suppressed the kynurenine pathway and showed promising anti-cancer efficacy, with favorable pharmacologic profiles.

ITOC2 – 013. In vitro kynurenine modulation by novel dual-acting and selective TDO and IDO inhibitors

Indoleamine 2,3-dioxygenase 1 (IDO1), indoleamine 2,3-dioxygenase 2 (IDO2), and tryptophan 2,3-dioxygenase (TDO) initiate the first step of the kynurenine pathway (KP), leading to the transformation of l-tryptophan (Trp) into l-kynurenine (Kyn) and other downstream metabolites. Kyn is known as an endogenous ligand of the aryl hydrocarbon receptor (AhR). Activation of AhR through TDO-derived Kyn is a novel mechanism to support tumor growth in gliomas. However, the role of IDO1 and IDO2 in this mechanism is still unknown. Herein, by using clinical samples, we found that the expression and activity of IDO1 and/or TDO (IDO1/TDO) rather than IDO2 were positively correlated with the pathologic grades of gliomas. The expression of IDO1/TDO rather than IDO2 was positively correlated with the Ki67 index and overall survival. The expression of IDO1/TDO was positively correlated with the expression of aquaporin 4 (AQP4), implying the potential involvement of IDO1/TDO in glioma cell motility. Mechanistically, we found that IDO1/TDO accounted for the release of Kyn, which activated AhR to promote cell motility via the Kyn–AhR–AQP4 signaling pathway in U87MG glioma cells. RY103, an IDO1/TDO dual inhibitor, could block the IDO1/TDO–Kyn–AhR–AQP4 signaling pathway and exert anti-glioma effects in GL261 orthotopic glioma mice. Together, our results showed that the IDO1/TDO–Kyn–AhR–AQP4 signaling pathway is a new mechanism underlying the malignancy of gliomas, and suggest that both IDO1 and TDO might be valuable therapeutic targets for gliomas.

Introduction: Tumor immune escape mechanisms have been established as suitable targets for cancer therapy. Among these, tryptophan catabolism plays a central role in creating an immunosuppressive environment, leading to tolerance to potentially immunogenic tumor antigens. Tryptophan catabolism is initiated by either indoleamine 2,3-dioxygenase (IDO1/2) or tryptophan 2,3-dioxygenase 2 (TDO2), resulting in biostatic tryptophan starvation and l-kynurenine production. Recent literature has shown that IDO1 and TDO2 are expressed in multiple tumors, including solid tumors and play key roles in tumor progression other than immune escape. It has also been shown that IDO1 and TDO2 play distinct roles in driving the downstream effectors suggesting that their roles are perhaps non-redundant. Therefore, we developed series of novel small molecule modulators against IDO1 and TDO2 to understand their role in disease biology for multiple indications including cancer, depression and autoimmune disorders. Methods: Rational design approaches were used to design novel IDO1 and TDO2 specific modulators; Potency of these inhibitors was assessed in the in vitro assays using purified IDO1 and TDO2 enzymes and in IDO1 and TDO2 over-expressing HEK293T cells by measuring the formation of kynurenine. Results: In the in vitro biochemical assay using purified human TDO2, one of the NCEs from inhibitor series showed an IC50 of 0.09 μM against TDO2. Against purified human IDO1, this compound was inactive up to 30 μM, thereby showing >300-fold selectivity against IDO1. This TDO2 inhibitory activity translated well in TDO2-overexpressing HEK293 cells and, these NCEs inhibited kynurenine formation with an EC50 of ~2.5 μM. A second chemical series showed comparable dual inhibition of IDO1 and TDO2 activity. One of the molecules from this series showed IC50 of 0.2 and 0.08 μM in the biochemical assay and 1.7 and 0.8 μM in the cell based assay against IDO1 and TDO2, respectively. In addition, another series of NCEs showed strong activation of IDO1 and TDO2 activity. As compared to untreated control, formation of kynurenine was increased in a dose-dependent manner as observed by increase in fluorescence up to of 3-10 folds. One of the compounds from this series showed an EC50 of ~20- 30 μM in the biochemical assay as well as in 293T-based assay. Further mechanistic studies to understand the immune modulatory activity of these selective TDO2 modulators is underway. These tool compounds are being further optimized for potency and ADME properties to be developed as potential drug candidates. Conclusion: To our knowledge such IDO1 and TDO2 specific small molecule activators have not been reported earlier. Therefore, these activators and inhibitors would serve as useful tool compounds in understanding the specific role(s) of IDO1 and TDO2 in disease biology and would also provide us the opportunity to target this pathway for various diseases, including cancer. Citation Format: Shivani Rao Garapaty, Dhanalakshmi Sivanandhan, Guru Pavan Kumar Seerapu, Surendra Naidu, Shalini Chakelam, Pradeep Nagaraj, Reshma Das, Saravanan Vadivelu, Pravin Iyer, Chandrika Mulakala, Kannan Murugan, Somnath Mondal, Anjali Gautam, Saravanan Kandan, Manish Kumar Thakur, Sridharan Rajagopal, Sriram Rajagopal. Small molecule modulators to understand the role of IDO1 and TDO2 in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5578. doi:10.1158/1538-7445.AM2017-5578

To assess the role of the kynurenine pathway in the pathology of Alzheimer's disease (AD), the expression and localization of key components of the kynurenine pathway including the key regulatory enzyme tryptophan 2,3 dioxygenase (TDO), and the metabolites tryptophan, kynurenine, kynurenic acid, quinolinic acid and picolinic acid were assessed in different brain regions of triple transgenic AD mice. The expression and cell distribution of TDO and quinolinic acid, and their co-localization with neurofibrillary tangles and senile β amyloid deposition were also determined in hippocampal sections from human AD brains. The expression of TDO mRNA was significantly increased in the cerebellum of AD mouse brain. Immunohistochemistry demonstrated that the density of TDO immuno-positive cells was significantly higher in the AD mice. The production of the excitotoxin quinolinic acid strongly increased in the hippocampus in a progressive and age-dependent manner in AD mice. Significantly higher TDO and indoleamine 2,3 dioxygenase 1 immunoreactivity was observed in the hippocampus of AD patients. Furthermore, TDO co-localizes with quinolinic acid, neurofibrillary tangles-tau and amyloid deposits in the hippocampus of AD. These results show that the kynurenine pathway is over-activated in AD mice. This is the first report demonstrating that TDO is highly expressed in the brains of AD mice and in AD patients, suggesting that TDO-mediated activation of the kynurenine pathway could be involved in neurofibrillary tangles formation and associated with senile plaque. Our study adds to the evidence that the kynurenine pathway may play important roles in the neurodegenerative processes of AD.